Ovalbumin titration

With several other proteins, such as bovine serum albiunin (Tanford and Roberts, 1952), lysozyme (Tanford and Wagner, 1954), and/3-lacto-globulin (Tanford and Swanson, 1957), pK shifts of the phenolic OH groups of tyrosine residues are observed, but these are of a qualitatively different nature. Thus, the tyrosines of any one of these proteins cannot be readily differentiated into a normal and an abnormal variety, since the spectrophotometric titration data for these proteins are reversible and fall on single smooth curves, in contrast to the situation with RNase. On the other hand, the tyrosine residues of ovalbumin show comparable behavior to the three abnormal tyrosine groups of RNase (Crammer and Neuberger, 1943). About 2 of the total of 9 tyrosine residues appear to titrate normally, but the remainder are not titrated up to pH 12. At pH 13, these anomalous tyrosines become titratable, and this is accompanied by the irreversible denaturation of the ovalbumin molecule. 

Table VIII shows that the volume changes for ovalbumin between pH 5.2 and 6.8 are especially low. This cannot be explained in a simple way, e.g., by the fact that there are phosphate groups in ovalbumin which titrate in this range of pH (because only four of the twenty groups which are titrated are phosphate groups). There is also no evidence that ovalbumin undergoes a significant conformational change between pH 5 and pH 7.

The very first electrometric titration curve of a protein to be reported in the literature is a study of ovalbumin (Bugarszky and Liebermann, 1898). 

The first detailed analysis of a protein titration curve, according to the semiempirical treatment used for most of the titration curves reviewed in this paper, also involves ovalbumin (Cannan el al., 1941). The first discovery of phenolic groups inaccessible to titration was again made with this protein (Crammer and Neuberger, 1943). 

The study by Martin et al. is of interest not only for the rationalization of the electrometric and spectrophotometric measurements in terms of the microconstants, but also because the spectrophotometric titration of tyrosine relates so closely to similar studies in proteins. In particular, the multiple H+-equilibria of tjnrosine result from the close juxtaposition of amino and phenolic groups in the same molecule under these circumstances the ionizations are mutually interacting. We suggest that some of the anomahes seen in t3Tosyl ionization in proteins may arise in a similar fashion, but in terms of magnitude, this mechanism clearly cannot account for such anomalous tjn-osyl groups as those seen in ribonuclease or ovalbumin. 

Figure 13 shows these changes in extinction for the NBS titration of tryptophan in bovine serum albumin (Ramachandran and Witkop, 1959), which is dissolved in 10.0 M urea solution in order to make the tryptophan units accessible. Another convenient way of picturing the changes in extinction is shown in Fig. 14 (Peters, 1959). Here one recognizes at a glance that ribonuclease contains no tryptophan. Based on a value of 2.8 X 10 for the amplitude in drop of molar absorption of free tryptophan it was concluded that human serum albumin (HSA) contains one, bovine serum albumin (BSA) two, and ovalbumin probably four rather than three tryptophan units.

This inference is not, however, inconsistent with the demonstration by Stein-hardt, Fugitt, and Harris (1941) that certain other monovalent anions do influence the acid titration of ovalbumin. 

For human serum albumin Tanford (1950) found by spectrophotometry that the ionization of the tyrosine hydroxyl groups was completely reversible up to pH 12. Measurements at the wavelength of the tyrosine anion maximum (2930 A.), uncorrected for the small tryptophan contribution, gave a pK of 11.7 for this process. Both the ultraviolet absorption and titration data for this protein could be quantitatively interpreted on the basis of complete freedom of all the 18 tyrosine hydroxyl groups in the molecule to ionize. In this respect human serum albumin thus resembles insulin and not ovalbumin. 

Next, it is worth while comparing the data from titration curves with those from electrophoresis. Here again the best investigations have been made on corpuscular proteins. A beautiful example is to be found in the work of Cannan, Kibrick and Palmer on the titration and that of Longsworth on the electrophoresis of ovalbumin. 

Turbidimetric titration curves were obtained for lysozyme in the absence (Fig. 16.6) and presence (Fig. 16.7) of ovalbumin the critical pH values are... 

In the titration curves of lysozyme in the presence of ovalbumin, ovalbumin only weakly interfered with the complexation of lysozyme and PAA. However, in the egg white system, only 3.4% of the protein is lysozyme. Most of the proteins have net charges opposite to that of lysozyme below the isoelectric point of lysozyme, pH 10.7. The likelihood of interactions among the proteins shifting the critical pH is much greater Fig. 16.8 shows this to occur for all but the highest MW. Why the critical pH of MW 4000000 PAA was not much affected (compare Figs. 16.6 and 16.8) is not understood. However, the absence of a shift is an indication of selective removal of the lysozyme such behavior is desirable in trying to fractionate proteins by precipitation. 

For turbidimetric titrations in the binary system, ovalbumin lowers the critical pH of lysozyme less than 0.1 pH unit perhaps as the result of weak interference with PAA/lysozyme complexation. For turbidimetric titrations of single proteins and mixtures, larger molecular weight PAA gave higher critical pH values. In contrast, increased ionic strength lowers the critical pH value as a result of electrostatic screening. 

Another phosphoprotein that can bind metal ions is ovalbumin, the major protein found in hen egg whites (Taborsky, 1974). The protein is phosphorylated at two positions, serine-68 and -344 (Nisbet et ai, 1981). These two residues are well resolved in a P-NMR spectrum, both shift as a function of pH, with p values and Hill coefficients identical to those of standards titrated under the same conditions (Vogel and Bridger, 1982c). The residues could be assigned by phosphatase treatment (Fig. 12). The resonance remaining after digestion with phosphatase corresponds to Ser-68, whereas the phosphatase-sensitive (more upheld) peak is SerP-344. 

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